Use of AgNOR staining to determine the effect of metoclopramide on neural tube development in early chick embryos

Abstract

Nausea and vomiting during pregnancy are common problems and prolonged pharmacological treatment often is needed; however, the teratogenic effects of anti-emetic drugs on neural tube (NT) development are not clear. We investigated the effects of different doses of metoclopramide on NT development in 48 and 72 h chick embryos using an argyrophilic nucleolar organizing region (AgNOR) staining method. We used 150 fertile, specific pathogen-free eggs incubated for 28 h, then randomly divided into five equal groups: group A, sham control was administered 0.9% saline; groups B - E were administered 0.15 mg/egg, 0.3 mg/egg, 0.6 mg/egg and 1.2 mg/egg, respectively. Half of the eggs in each group were taken from the incubator at 48 h incubation and the other half at 72 h incubation. After incubation, eggs were opened, embryos were dissected from their membranes, fixed with 10% formalin and examined by light microscopy. The NT status, i.e., open or closed, and somite number, crown-rump length, morphological features and gross developmental abnormalities were recorded. Excised embryos were sectioned and stained using hematoxylin and eosin or the AgNOR procedure and examined for morphology and histopathology. Delayed NT closure was observed in all 48 h drug exposed embryos, but in the 72 h groups, this occurred only in high-dose groups. Somite number was reduced significantly in groups C - E compared to the control group. Crown-rump length was decreased in both 48 and 72 h embryos. We found a decreased total AgNOR area:nuclear area ratio in 48 and 72 h embryos of all experimental groups. We found that metoclopramide delayed NT closure in chick embryos in a dose-dependent manner.